Development of a novel and affordable point-of-care kit for rapid detection of urea and glucose adulteration in cow milk

Abstract

The increasing global population has raised the demand for cow milk, leading to its adulteration with harmful substances, including urea and glucose, that cause damage to humans when consumed regularly. Hence, this study started with predicting urea and glucose toxicity using ProTox-III software, wherein the results revealed that urea belongs to class IV with an LD₅₀ value of 6350 mg kg⁻¹ and glucose belongs to class VI with an LD₅₀ value of 23 000 mg kg⁻¹. Then, a qualitative colorimetric kit and Fourier-transform infrared (FTIR) spectroscopy were used for the preliminary detection of urea and glucose in cow milk. The colorimetric kit confirmed the presence of urea and glucose by changing the sample colour. Based on these results, a point-of-care (PoC) kit was developed for urea and glucose detection in cow milk. The enzyme immobilization technique was used to coat urease and glucose oxidase/peroxidase on polystyrene strips to make PoC strips. The biochemical methods of the Berthelot assay and glucose oxidase/peroxidase (GOD/POD) assay were used to detect urea and glucose, respectively. The lowest detection limits of the developed microassay kit for urea and glucose were 1.5 and 3 μg from 300 μg of cow milk. The shelf life of the urease immobilized strip was ∼30 days, with 15 times the reusability of a single well, and for the GOD/POD immobilized strip it was ∼15 days, with 7 times the reusability, each with a detection efficiency of 85–90%. The strips provided results in ten minutes and were easily portable for on-site adulteration detection.